Rank ligand-binding polypeptides

Abstract

The present invention relates to a polypeptide having an amino acid sequence that differs from and is at least 70% identical to the amino acid sequence of hRANK, and which has a binding affinity to RANKL that is at least as high as the binding affinity of hRANK to RANKL, as determined by the functional competition assay described herein.

1 . A polypeptide having an amino acid sequence that differs from and is at least about 70% identical to the amino acid sequence of hRANK, and which has a binding affinity to RANKL that is at least as high as the binding affinity of hRANK to RANKL, as determined by the functional competition assay described herein.  
     
     
         2 . The polypeptide of  claim 1 , which has an increased binding affinity to RANKL compared to the binding affinity of hRANK in the functional competition assay.  
     
     
         3 . The polypeptide of  claim 1  or  2 , having an amino acid sequence that is at least about 75% identical to the amino acid sequence of hRANK, e.g. at least about 80%, 85%, 90% or 95%.  
     
     
         4 . The polypeptide of any of claims  1 - 3 , having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         5 . The polypeptide of  claim 4 , wherein the non-polypeptide moiety is selected from the group consisting of polymer molecules, oligosaccharide moieties, lipophilic compounds and organic derivatizing agents.  
     
     
         6 . The polypeptide of  claim 5 , wherein the non-polypeptide moiety is a PEG molecule.  
     
     
         7 . The polypeptide of any of claims  1 - 6 , which has an increased functional in vivo half-life and/or serum half-life compared to hRANK.  
     
     
         8 . A polypeptide having an amino acid sequence that differs from and is least about 70% identical to the amino acid sequence of hOPG, and which has a binding affinity to RANKL that is at least as high as the binding affinity of hOPG to RANKL, as determined by the functional competition assay described herein.  
     
     
         9 . The polypeptide of  claim 8 , which has an increased binding affinity to RANKL compared to the binding affinity of hOPG in the functional competition assay.  
     
     
         10 . The polypeptide of  claim 8  or  9 , having an amino acid sequence that is at least about 75% identical to the amino acid sequence of hOPG, e.g. at least about 80%, 85%, 90% or 95%.  
     
     
         11 . The polypeptide of any of claims  8 - 10 , having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         12 . The polypeptide of  claim 11 , wherein the non-polypeptide moiety is selected from the group consisting of polymer molecules, oligosaccharide moieties, lipophilic compounds and organic derivatizing agents.  
     
     
         13 . The polypeptide of  claim 12 , wherein the non-polypeptide moiety is a PEG molecule.  
     
     
         14 . The polypeptide of any of claims  8 - 13 , which has an increased functional in vivo half-life and/or serum half-life compared to hOPG.  
     
     
         15 . A polypeptide having an amino acid sequence that is at least 40% identical to the amino acid sequence of hRANK and at least 40% identical to the amino acid sequence of hOPG, and which has a binding affinity to RANKL at least as high as the binding affinity of hRANK and hOPG to RANKL, as determined by the functional competition assay described herein.  
     
     
         16 . The polypeptide of  claim 15 , which has an increased binding affinity to RANKL compared to the binding affinity of hRANK and hOPG in the functional competition assay.  
     
     
         17 . The polypeptide of  claim 15  or  16 , having an amino acid sequence that is at least about 45% identical to the amino acid sequence of hRANK and/or hOPG, e.g. at least about 50%, 55%, 60%, 65%, 70%, 75% or 80%.  
     
     
         18 . The polypeptide of any of claims  15 - 17 , having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         19 . The polypeptide of  claim 18 , wherein the non-polypeptide moiety is selected from the group consisting of polymer molecules, oligosaccharide moieties, lipophilic compounds and organic derivatizing agents.  
     
     
         20 . The polypeptide of  claim 19 , wherein the non-polypeptide moiety is a PEG molecule.  
     
     
         21 . A chimeric polypeptide comprising a RANK backbone wherein at least one amino acid residue of the RANK backbone has been substituted with the corresponding amino acid residue from an OPG polypeptide as determined by a sequence alignment.  
     
     
         22 . The chimeric polypeptide of  claim 21 , wherein at least 2, preferably at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, such as up to about 15 or 20 amino acid residues of the RANK backbone have been substituted with the corresponding amino acid residues from the OPG polypeptide.  
     
     
         23 . The chimeric polypeptide of  claim 21  or  22 , wherein at least one amino acid residue substitution is in the TNF receptor-like domain, preferably in a ligand binding domain.  
     
     
         24 . The chimeric polypeptide of any of claims  21 - 23 , wherein the RANK backbone is hRANK.  
     
     
         25 . The chimeric polypeptide of any of claims  21 - 24 , which has an improved binding affinity to RANKL compared to the binding affinity of hRANK to RANKL, as determined by the functional competition assay described herein.  
     
     
         26 . The chimeric polypeptide of any of claims  21 - 25 , having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         27 . A chimeric polypeptide comprising an OPG backbone wherein at least one amino acid residue of the OPG backbone has been substituted with the corresponding amino acid residue from a RANK polypeptide as determined by a sequence alignment.  
     
     
         28 . The chimeric polypeptide of  claim 27 , wherein at least 2, preferably at least 3, e.g. at least 4, 5, 6, 7, 8, 9 or 10, such as up to about 15 or 20 amino acid residues of the OPG backbone have been substituted with the corresponding amino acid residues from the RANK polypeptide.  
     
     
         29 . The chimeric polypeptide of  claim 27  or  28 , wherein at least one amino acid residue substitution is in the TNFR-like domain, preferably in a ligand binding domain.  
     
     
         30 . The chimeric polypeptide of any of claims  27 - 29 , wherein the OPG backbone is hOPG.  
     
     
         31 . The chimeric polypeptide of any of claims  27 - 30 , which has an improved binding affinity to RANKL compared to the binding affinity of hOPG to RANKL, as determined by the functional competition assay described herein.  
     
     
         32 . The chimeric polypeptide of any of claims  27 - 31 , having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         33 . A method for obtaining a nucleic acid encoding a recombinant polypeptide having RANKL binding activity, the method comprising: 
 (a) creating a library of recombinant polynucleotides encoding one or more recombinant RANK polypeptides; and    (b) screening the library to identify a recombinant polynucleotide encoding a recombinant polypeptide with a binding affinity to RANKL at least as high as the binding affinity of hRANK to RANKL.    
     
     
         34 . The method of  claim 34 , comprising selecting at least one recombinant polynucleotide encoding a recombinant polypeptide with a binding affinity to RANKL higher than the binding affinity of hRANK to RANKL.  
     
     
         35 . The method of  claim 34 , wherein said library is created by subjecting a plurality of parental polynucleotides to site-directed or random mutagenesis to produce at least one recombinant RANK polynucleotide encoding said improved recombinant polypeptide  
     
     
         36 . The method of  claim 33 , wherein said library is created by shuffling a plurality of parental polynucleotides to produce at least one recombinant RANK polynucleotide encoding said improved recombinant polypeptide.  
     
     
         37 . The method of  claim 36 , wherein said parental polynucleotides are homologous.  
     
     
         38 . The method of  claim 36 , wherein said parental polynucleotides are shuffled in a plurality of cells selected from prokaryotes and eukaryotes, e.g. in eukaryotic cells selected from bacteria, yeast, fungi and mammalian cells.  
     
     
         39 . The method of  claim 36 , further comprising: 
 (c) recombining at least one distinct or improved recombinant polynucleotide with a further polynucleotide encoding a polypeptide with RANKL binding affinity, which further polynucleotide is identical to or different from one or more of said plurality of parental polynucleotides, to produce a library of recombinant polynucleotides;    (d) screening said library to identify at least one further distinct or improved recombinant polynucleotide encoding a RANKL binding polypeptide that exhibits a further improvement or distinct property compared to a polypeptide encoded by said plurality of parental polynucleotides; and, optionally,    (e) repeating (c) and (d) until said resulting further distinct or improved recombinant polynucleotide shows an additionally distinct or improved property.    
     
     
         40 . The method of  claim 36 , wherein said recombinant polynucleotides are present in one or more cells selected from bacterial, yeast, fungal and mammalian cells, and said method comprises: 
 pooling multiple separate polynucleotides;    screening said resulting pooled polynucleotides to identify an improved recombinant polynucleotide encoding a polypeptide that exhibits an improved binding affinity to RANKL compared to a polypeptide encoded by a non-recombinant activity polynucleotide; and    cloning said improved recombinant nucleic acid.    
     
     
         41 . The method of  claim 40 , further comprising transducing said improved polynucleotide into a member selected from a prokaryote and a eukaryote.  
     
     
         42 . The method of  claim 36 , wherein said shuffling of a plurality of parental polynucleotides comprises at least one shuffling technique selected from family gene shuffling, individual gene shuffling and in silico shuffling.  
     
     
         43 . A library of recombinant polynucleotides encoding at least one polypeptide with binding affinity to RANKL, wherein said library is made by the method of any of claims  33 - 42 .  
     
     
         44 . The library of  claim 43 , wherein polypeptides encoded by said recombinant polynucleotides are displayed on the surface of phage, bacteria cells, yeast cells or mammalian cells.  
     
     
         45 . A nucleic acid encoding a polypeptide with binding affinity to RANKL, wherein said nucleic acid is prepared by the method of any of claims  33 - 42 .  
     
     
         46 . A nucleic acid shuffling mixture, comprising: at least three homologous DNAs, each of which is derived from a polynucleotide encoding a polypeptide selected from a parent RANK polypeptide, a polypeptide fragment having RANKL binding affinity, and combinations thereof.  
     
     
         47 . The nucleic acid shuffling mixture of  claim 46 , wherein said at least three homologous DNAs are present in cell culture or in vitro.  
     
     
         48 . A polypeptide having RANKL binding affinity encoded by a nucleic acid produced by the method of any of claims  33 - 42 .  
     
     
         49 . A method for obtaining a nucleic acid encoding a recombinant polypeptide having RANKL binding activity, the method comprising: 
 (a) creating a library of recombinant polynucleotides encoding one or more recombinant OPG polypeptides; and    (b) screening the library to identify a recombinant polynucleotide encoding a recombinant polypeptide with a binding affinity to RANKL at least as high as the binding affinity of hOPG to RANKL.    
     
     
         50 . The method of  claim 49 , comprising selecting at least one recombinant polynucleotide encoding a recombinant polypeptide with a binding affinity to RANKL higher than the binding affinity of hOPG to RANKL.  
     
     
         51 . The method of  claim 49 , wherein said library is created by subjecting a plurality of parental polynucleotides to site-directed or random mutagenesis to produce at least one recombinant OPG polynucleotide encoding said improved recombinant polypeptide  
     
     
         52 . The method of  claim 49 , wherein said library is created by shuffling a plurality of parental polynucleotides to produce at least one recombinant OPG polynucleotide encoding said improved recombinant polypeptide.  
     
     
         53 . The method of  claim 52 , wherein said parental polynucleotides are homologous.  
     
     
         54 . The method of  claim 52 , wherein said parental polynucleotides are shuffled in a plurality of cells selected from prokaryotes and eukaryotes, e.g. in eukaryotic cells selected from bacteria, yeast, fungi and mammalian cells.  
     
     
         55 . The method of  claim 52 , further comprising: 
 (c) recombining at least one distinct or improved recombinant polynucleotide with a further polynucleotide encoding a polypeptide with RANKL binding affinity, which further polynucleotide is identical to or different from one or more of said plurality of parental polynucleotides, to produce a library of recombinant polynucleotides;    (d) screening said library to identify at least one further distinct or improved recombinant polynucleotide encoding a RANKL binding polypeptide that exhibits a further improvement or distinct property compared to a polypeptide encoded by said plurality of parental polynucleotides; and, optionally,    (e) repeating (c) and (d) until said resulting further distinct or improved recombinant polynucleotide shows an additionally distinct or improved property.    
     
     
         56 . The method of  claim 52 , wherein said recombinant polynucleotides are present in one or more cells selected from bacterial, yeast, fungal and mammalian cells, and said method comprises: 
 pooling multiple separate polynucleotides;    screening said resulting pooled polynucleotides to identify an improved recombinant polynucleotide encoding a polypeptide that exhibits an improved binding affinity to RANKL compared to a polypeptide encoded by a non-recombinant activity polynucleotide; and    cloning said improved recombinant nucleic acid.    
     
     
         57 . The method of  claim 56 , further comprising transducing said improved polynucleotide into a member selected from a prokaryote and a eukaryote.  
     
     
         58 . The method of  claim 52 , wherein said shuffling of a plurality of parental polynucleotides comprises at least one shuffling technique selected from family gene shuffling, individual gene shuffling and in silico shuffling.  
     
     
         59 . A library of recombinant polynucleotides encoding at least one polypeptide with binding affinity to RANKL, wherein said library is made by the method of any of claims  49 - 58 .  
     
     
         60 . The library of  claim 58 , wherein polypeptides encoded by said recombinant polynucleotides are displayed on the surface of phage, bacteria cells, yeast cells or mammalian cells.  
     
     
         61 . A nucleic acid encoding a polypeptide with binding affinity to RANKL, wherein said nucleic acid is prepared by the method of any of claims  49 - 58 .  
     
     
         62 . A nucleic acid shuffling mixture, comprising: at least three homologous DNAs, each of which is derived from a polynucleotide encoding a polypeptide selected from a parent OPG polypeptide, a polypeptide fragment having RANKL binding affinity, and combinations thereof.  
     
     
         63 . The nucleic acid shuffling mixture of  claim 62 , wherein said at least three homologous DNAs are present in cell culture or in vitro.  
     
     
         64 . A polypeptide having RANKL binding affinity encoded by a nucleic acid produced by the method of any of claims  49 - 58 .  
     
     
         65 . A polypeptide conjugate exhibiting RANKL-binding activity, comprising a RANK polypeptide that differs from wild-type human RANK in that at least one amino acid residue acid residue comprising an attachment group for a non-polypeptide moiety has been introduced or removed, and having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         66 . The polypeptide conjugate of  claim 65 , wherein the RANK polypeptide is a RANK variant as defined in any of claims  1 - 7  or  21 - 26  or encoded by a nucleic acid produced by the method of any of claims  33 - 42 .  
     
     
         67 . A polypeptide conjugate exhibiting RANKL-binding activity, comprising an OPG polypeptide that differs from wild-type human OPG in that at least one amino acid residue acid residue comprising an attachment group for a non-polypeptide moiety has been introduced or removed, and having at least one non-polypeptide moiety bound to an attachment group of the polypeptide.  
     
     
         68 . The polypeptide conjugate of  claim 67 , wherein the OPG polypeptide is an OPG variant as defined in any of claims  8 - 14  or  27 - 32  or encoded by a nucleic acid produced by the method of any of claims  49 - 58 .  
     
     
         69 . An oligomeric fusion protein comprising at least two RANK monomers, at least two OPG monomers, or at least one RANK monomer and at least one OPG monomer, wherein at least one monomer of the fusion protein is a RANK and/or OPG variant as defined in any of claims  1 - 32  or encoded by a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 .  
     
     
         70 . The fusion protein of  claim 69 , wherein the monomers are joined by a peptide bond or a peptide linker, or by a PEG molecule.  
     
     
         71 . The fusion protein of  claim 69 , comprising at least one RANKL-binding monomeric fusion protein, wherein said monomeric fusion protein is produced as a protein fused in frame with an immunoglobulin Fc polypeptide or a GCN4 leucine zipper.  
     
     
         72 . A composition comprising a polypeptide according to any of claims  1 - 31  or  65 - 71  or encoded by a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 , and at least one pharmaceutically acceptable carrier or excipient.  
     
     
         73 . Use of a polypeptide according to any of claims  1 - 31  or  65 - 71  or encoded by a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 , or a composition according to  claim 72 , as a pharmaceutical.  
     
     
         74 . Use of a polypeptide according to any of claims  1 - 31  or  65 - 71  or encoded by a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 , or a composition according to  claim 72 , for the preparation of a medicament for the prevention or treatment of osteoporosis or other bone diseases or other diseases associated with binding of RANKL to the RANK receptor.  
     
     
         75 . A method for preventing or treating osteoporosis or other bone diseases or other diseases associated with binding of RANKL to the RANK receptor, the method comprising administering to a patient in need thereof an effective amount of a polypeptide according to any of claims  1 - 32  or  65 - 71  or encoded by a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 , or a composition according to  claim 72 .  
     
     
         76 . An expression vector comprising a nucleic acid produced by the method of any of claims  33 - 42  or  49 - 58 .  
     
     
         77 . A host cell comprising an expression vector according to  claim 76 .  
     
     
         78 . A method for producing a polypeptide having binding affinity to RANKL, comprising culturing a host cell according to  claim 77  under conditions conducive for expression of the polypeptide, and recovering the polypeptide.  
     
     
         79 . The method of  claim 78 , wherein a) the polypeptide comprises at least one N- or O-glycosylation site and the host cell is a eukaryotic host cell capable of in vivo glycosylation, and/or b) the polypeptide is subjected to conjugation to a non-polypeptide moiety in vitro.  
     
     
         80 . The chimeric polypeptide of  claim 21 , comprising all or part of at least one TNF receptor-like domain of OPG as defined in FIG. 4B.  
     
     
         81 . The chimeric polypeptide of  claim 80 , wherein said part comprises at least one ligand binding subsequence of OPG comprising at least three amino acid residues as defined in FIG. 4B.  
     
     
         82 . The chimeric polypeptide of  claim 27 , comprising all or part of at least one TNF receptor-like domain of RANK as defined in FIG. 4B.  
     
     
         83 . The chimeric polypeptide of  claim 82 , wherein said part comprises at least one ligand binding subsequence of RANK comprising at least three amino acid residues as defined in FIG. 4B.  
     
     
         84 . A method for obtaining a nucleic acid encoding a recombinant polypeptide having a desired RANKL binding activity, the method comprising: 
 (a) providing a polynucleotide encoding a recombinant chimeric polypeptide comprising at least one ligand binding sequence from an OPG domain and at least one ligand binding sequence from a RANK domain;    (b) subjecting said polynucleotide to mutagenesis to create a library of recombinant polynucleotides encoding one or more recombinant chimeric polypeptides; and    (c) screening the library to identify a recombinant polynucleotide encoding a recombinant polypeptide with a desired binding affinity to RANKL.    
     
     
         85 . The method of  claim 84 , wherein said recombinant chimeric polypeptide in (a) comprises at least one OPG domain and at least one RANK domain.  
     
     
         86 . The method of  claim 84  or  85 , wherein mutagenesis is performed using at least one of site-directed mutagenesis, random mutagenesis and shuffling.  
     
     
         87 . A polypeptide having an amino acid sequence that is least about 70% identical to the amino acid sequence of hOPG(22-194) and wherein one or more of the amino acid residues selected from T71, K108, R111, and T154 have been substituted with a different amino acid residue.  
     
     
         88 . A polypeptide comprising the amino acid sequence hOPG(22-194) wherein one or more of the amino acid residues selected from T71, K108, R111, and T154 have been substituted with a different amino acid residue.  
     
     
         89 . The polypeptide of  claim 87  or  88  wherein T71 has been substituted with A.  
     
     
         90 . The polypeptide of any one of claims  87 - 89  wherein K108 has been substituted with N.  
     
     
         91 . The polypeptide of any one of claims  87 - 90  wherein R111 has been substituted with W.  
     
     
         92 . The polypeptide of any one of claims  87 - 91  wherein T154 has been substituted with L.  
     
     
         93 . The polypeptide of any one of claims  87 - 92  whereint the polypeptide is selected from the group comprising T71A,K108N-hOPG(22-194), R111W-hOPG(22-194), K108M,R111W-hOPG(22-194), and T154L-hOPG(22-194).